Rumen fermentation improving agent

ABSTRACT

The present invention provides a rumen fermentation improving agent, comprising cashew nut shell liquid.

TECHNICAL FIELD

The present invention relates to a rumen fermentation improving agent, acomposition for a feed, and a feed each containing cashew nut shellliquid, and a breeding method for a ruminant or the like using the same.

BACKGROUND ART

In ruminants such as cattle and sheep, a feed is digested and fermentedby microorganisms in their rumens, and the ruminants live by utilizingthe fermentation products. Therefore, it is a loss of energy efficiencywhen methane is generated from the rumens. Further, because methane is agreenhouse gas that influences the global warming, it is important toreduce the amount of methane produced in the rumens of the ruminants.

A methanogen in a rumen reduces carbon dioxide by utilizing hydrogen tothereby produce methane. The contribution ratio of methane to the globalwarming is second highest after carbon dioxide, and it is consideredthat methane emitted from ruminants accounts for 15 to 20% of the totalemission of methane.

In 1940s, it was found that the growth of a domestic animal is promotedby adding a small amount of an antibiotic to a domestic animal feed.Since then, the practice of adding the antibiotic to the domestic animalfeed has been performed widely as means for promoting the growth of thedomestic animal.

It is considered that the antibiotic exhibits an action of promoting thegrowth by its effects of (1) prevention of pathogenic bacterialinfection of a domestic animal, (2) improvement in metabolism, and (3)suppression of proliferation of harmful enterobacteria, but the detailsthereof are still unclear. On the other hand, as a result of addingantibiotics to a feed, the antibiotics are distributed widely in theenvironment, and accordingly, an appearance of antibiotic-resistantbacteria has become a social problem. In recent years, the addition ofan antibiotic to a feed has been regulated strictly, and in Europe, theuse of the antibiotic for promoting the growth was banned by January2006. Further, there is a strong demand from producers for livestockproducts that are bred without using an antibiotic, and hence, the needof an alternative to the antibiotic is growing.

Ionophores such as monensin, which are antibiotics, are widely used in afeed for a ruminant. Monensin exhibits a selective suppressing effectwith respect to a rumen microorganism and has, as a result, functions ofreducing methane production and promoting propionic acid production.Propionic acid has higher ATP production efficiency compared with othervolatile fatty acids, and hence, feed efficiency is improved owing tothe promotion of propionic acid production.

There is also desired the development of alternatives to monensin andthe like to be added to a feed for a ruminant. As the alternatives,there are studied an oil extracted from a plant (Non-patent Document 1),a vaccine against lactic acid-producing bacteria (Non-patent Document2), an egg yolk antibody against lactic acid-producing bacteria(Non-patent Document 3), and the like. However, those technologies arenot in practical use yet, because there are problems in that theireffects are not stable, the registration thereof as feeds is notpermitted, and the like. Further, there is also studied gluconic acid(Patent Document 1), but the amount of propionic acid production is notexemplified, so a methane production-suppressing effect is not known.

It is known that cashew nut shell liquid has an antibacterial action(Non-patent Document 4) and a coccidiosis-relieving action (PatentDocument 2). Further, as for the rumen function-improving effects of aruminant, an in vitro test result using anacardic acid (Non-patentDocument 5) is reported, but there is no disclosure on thereproducibility, application, and the optimum content on an actualanimal.

Patent Document 1: WO 01/028551

Patent Document 2: JP 08-231410 A

Non-patent Document 1: Benchaar et al., Can. J. Anim. Sci. 86, 91-96(2006)

Non-patent Document 2: Shu et al., FEMS Immunology & MedicalMicrobiology, 26(2), 153-158 (1999)

Non-patent Document 3: DiLorenzo et al., J. Anim. Sci., 84, 2178-2185(2006)

Non-patent Document 4: Muroi, H. et al., Bioorganic &Medicinal Chemistry12, 583-587 (2004)

Non-patent Document 5: Van Nevel C. J., et al., Applied Microbiology 21,365-366 (1971)

DISCLOSURE OF THE INVENTION

An object of the present invention is to promote the growth of aruminant and to improve feed efficiency by improving the rumenfermentation in the ruminant and contributing to suppressing thegeneration of greenhouse gas.

The inventors of the present invention have intensively studied in orderto solve the above problems, and as a result, the inventors have foundthat a feed containing cashew nut shell liquid suppresses methaneproduction and promotes propionic acid production in a rumen. Thus, thepresent invention has been accomplished.

That is, the present invention is as follows:

(1) A rumen fermentation improving agent, comprising cashew nut shellliquid.

(2) A composition for a feed for improving rumen fermentation,comprising the rumen fermentation improving agent according to (1).

(3) A feed for improving rumen fermentation, comprising the compositionfor a feed for improving rumen fermentation according to (2).

(4) A composition for a feed for improving rumen fermentation,comprising cashew nut shell liquid.

(5) A feed for improving rumen fermentation, comprising cashew nut shellliquid.

(6) A feed for improving rumen fermentation according to (3) or (5),which is for a ruminant.

(7) A method of breeding a ruminant, comprising allowing the ruminant toingest the feed for improving rumen fermentation according to any one of(3), (5), and (6).

(8) Use of cashew nut shell liquid in manufacturing a rumen fermentationimproving agent.

(9) Use of cashew nut shell liquid in manufacturing a composition for afeed for improving rumen fermentation.

(10) Use of cashew nut shell liquid in manufacturing a feed forimproving rumen fermentation.

(11) A method of improving rumen fermentation of a domestic animal,comprising administering cashew nut shell liquid to the domestic animal.

By allowing a ruminant to ingest a feed into which the rumenfermentation improving agent or the composition for a feed of thepresent invention is mixed, or by allowing a ruminant to ingest the feedof the present invention, methane production may be suppressed andpropionic acid production may be promoted. The feed of the presentinvention may be preferably used for breeding ruminants such as cattle,goats, and sheep.

BEST MODE FOR CARRYING OUT THE INVENTION

A rumen fermentation improving agent of the present invention includescashew nut shell liquid (CNSL). Here, rumen fermentation-improvingeffects include a methane production-suppressing effect and a propionicacid production-promoting effect in a rumen.

The cashew nut shell liquid to be used in the present invention is anoily liquid contained in the shell of the seed of a cashew nut tree(Anacardium occidentale L.). The cashew nut shell liquid contains, asthe components thereof, anacardic acids, cardanol, and cardol. Inparticular, in order to stably exhibit rumen fermentation-improvingeffects, cashew nut shell liquid in which anacardic acids are containedat a ratio of 40 mass % or more is preferred, and cashew nut shellliquid in which anacardic acids are contained at a ratio of 50 mass % ormore is more preferred. There are three kinds of anacardic acids: ananacardic acid having three double bonds at 8-, 11-, and 14-positions(hereinafter, referred to as 15:3); an anacardic acid having two doublebonds at 8- and 11-positions (hereinafter, referred to as 15:2); and ananacardic acid having one double bond at 8-position (hereinafter,referred to as 15:1). From the viewpoint of rumen function-improvingeffects, a ratio of (15:1):(15:2):(15:3) is preferably 80 to 100:85 to120:140 to 180 and more preferably 90 to 95:100 to 110:150 to 160.

On the other hand, in the case of using a pure anacardic acid, therearises a problem that the production cost becomes high from thefollowing reasons: the number of steps for producing the pure anacardicacid increases, because an anacardic acid needs to be subjected tosolvent extraction from cashew nut shells or cashew nut shell liquidfollowed by fractionation; an explosion-proof facility is required,because a solvent is employed; and a facility that enables to completeremoval of the solvent, because when the used solvent is remained in theanacardic acid, the anacardic acid cannot be used as a feed.

Therefore, the cashew nut shell liquid obtained by squeezing oil fromcashew nut shells is preferred from the viewpoint that the productioncost can be reduced. Further, a ratio of the above-mentioned anacardicacids, cardanol, and cardol is preferably 50 to 80:1 to 30:5 to 40 fromthe viewpoint of the rumen function-improving effects, and is morepreferably 55 to 65:5 to 20:10 to 30.

The anacardic acids, cardanol, and cardol each have an antioxidantaction, and thus it is considered that these components suppress theoxidation of each other. Therefore, the rumen fermentation improvingagent of the present invention containing cashew nut shell liquid canexhibit rumen fermentation-improving effects more stably than by usinganacardic acids, cardanol, or cardol alone, each having double bond(s)to be oxidized easily.

In general, the cashew nut shell liquid can be obtained by heating, butin the present invention, it is preferred that the cashew nut shellliquid be extracted under unheated conditions in order to preventanacardic acids, which are main constituent components, from beingdenatured by heat. For example, the cashew nut shell liquid can beobtained by an extracting method involving compressing the shell of acashew nut or a method described in JP 08-231410 A.

The cashew nut shell liquid used in the present invention may also be aliquid obtained by pulverizing/crushing the shell of a cashew nut.

For the cashew nut shell liquid used in the present invention, acommercially-available product which is not subjected to heat treatmentmay also be used.

The cashew nut shell liquid used in the present invention is preferablyunheated cashew nut shell liquid from the viewpoint of the stability ofactive ingredients.

The content of the cashew nut shell liquid in the rumen fermentationimproving agent of the present invention is, from the viewpoints ofeffects and costs, preferably 1 mass % to 100 mass %, more preferably 5mass % to 90 mass %, and still more preferably 10 mass % to 80 mass %,with respect to a total amount of the rumen fermentation improvingagent. When the content is 1 mass % or more, the rumen fermentationeffect can be exhibited effectively with a certain amount of the rumenfermentation improving agent. Further, a stock solution of cashew nutshell liquid may be directly orally administered.

The rumen fermentation improving agent of the present invention mayfurther contain, in addition to the cashew nut shell liquid, anarbitrary component(s) such as a component which is effective for thegrowth promotion of a ruminant, a nutritional supplement component, acomponent for enhancing the preservation stability. Examples of thearbitrary components include the followings: probiotics such asEnterococcus, Bacillus, and Bifidus; enzymes such as amylase and lipase;vitamins such as L-ascorbic acid, choline chloride, inositol, andfolate; minerals such as potassium chloride, iron citrate, magnesiumoxide, and phosphates; amino acids such as DL-alanine, DL-methionine,L-lysine; organic acids such as fumaric acid, butyric acid, lactic acid,acetic acid, and their salts; antioxidants such as ethoxyquin,dibutylhydroxytoluene, butylhydroxy anisole, ferulic acid, vitamine C,and vitamine E; fungicides such as calcium propionate; binders such ascarboxylmethyl cellurose (CMC), casein sodium, and sodium polyacrylate;emulsifiers such as lecithin, glycerin fatty acid ester and sorbitanfatty acid ester; pigments such as astaxanthin and canthaxanthin; andflavoring agents such as various esters, ethers, and ketones.

The formulation of the rumen fermentation improving agent of the presentinvention is not particularly limited, and the agent may be in anarbitrary form such as powder, liquid, solid, a tablet, a capsule, oremulsion. The rumen fermentation improving agent of the presentinvention can be produced by mixing cashew nut shell liquid and, ifrequired, an arbitrary component, and forming the mixture into apreparation. Note that, depending on the form of the formulation, thepulverized/crushed product of the above-mentioned cashew nut shell orthe cashew nut shell as it is without being subjected to any treatmentis mixed with another arbitrary component, and the mixture can be usedas the rumen fermentation improving agent of the present invention. Inaddition, without being mixed with another arbitrary component, thepulverized/crushed product as it is or the cashew nut shell as it is maybe used as the rumen fermentation improving agent, and the rumenfermentation improving agent itself may be used as a composition for afeed or a feed.

The composition for a feed of the present invention includes cashew nutshell liquid. Further, the composition for a feed of the presentinvention may also include the rumen fermentation improving agent. Thecontent of the cashew nut shell liquid in the composition for a feed ofthe present invention is, from the viewpoints of effects and costs,preferably 0.5 to 500,000 mass ppm, more preferably 5 to 100,000 massppm, and still more preferably 50 to 50,000 mass ppm with respect to adry mass of the composition for a feed. The composition for a feed ofthe present invention is mixed with another feed component used in petfoods and supplements for pets (hereinafter referred to as feed), tothereby produce a feed. The kind of the feed and the components otherthan the cashew nut shell liquid are not particularly limited. The feedis preferably for ruminants.

The feed of the present invention includes cashew nut shell liquid.Further, the feed of the present invention may also include thecomposition for a feed. The content of the cashew nut shell liquid inthe feed of the present invention is, from the viewpoints of effects andcosts, preferably 0.5 to 50,000 mass ppm, more preferably 5 to 10,000mass ppm, and still more preferably 50 to 5,000 mass ppm with respect toa dry mass of the feed. It should be noted that, in the rumen juice ofan actual ruminant, it is considered that the cashew nut shell liquid isdiluted about 10 fold, and hence, it is suggested that the cashew nutshell liquid be administered about 10 times the content of the effectivecontent in vitro, in the case of being used in vivo.

The feed of the present invention can be produced by adding cashew nutshell liquid or a composition for a feed including the cashew nut shellliquid as it is to a feed component and mixing the resultant. On thisoccasion, when a powdery or solid composition for a feed is used, theform of the composition for a feed may be modified into a liquid form ora gel form for the purpose of facilitating the mixing process. In thiscase, the following may be used as a liquid carrier: water; a vegetableoil such as soybean oil, rapeseed oil, or corn oil; or a water-solublepolymer compound such as a liquid animal oil, polyvinylalcohol,polyvinylpyrrolidone, or polyacrylic acid. Further, in order to keep theuniformity of the cashew nut shell liquid in the feed, the feed alsopreferably contains alginic acid, sodium alginate, xanthan gum, caseinsodium, gum arabic, guar gum, or water-soluble polysaccharides such astamarind seed polysaccharide.

The species of animals that ingest the feed of the present invention ispreferably ruminants. The feed of the present invention is suitable forbreeding, for example, ruminants such as cows, goats, and sheep. Theamount of feed ingested by an animal may be appropriately adjusteddepending on the animal's species, body weight, age, sex, healthcondition, feed component, etc. In this case, the amount of cashew nutshell liquid contained in the feed is preferably 0.005 to 500 g perruminant per day, more preferably 0.5 to 100 g per ruminant per day, andstill more preferably 0.5 to 50 g per ruminant per day.

Any method usually used may be adopted as a method of feeding animalsand a method of breeding animals depending on the species of animals.

EXAMPLES Example 1 Effects of Cashew Nut Shell Liquid (CNSL) on GasProduction and Production of Volatile Fatty Acids In Vitro (1) Sample

Cashew nut shell liquid (CNSL), which was extracted by compressingcashew nut shells, was obtained from Cashew Trading Co., Ltd. For aculture inoculum, there was used rumen juice (quadruple gauze filtrate)collected from a Holstein cow (fitted with rumen cannula) owned byExperiment Farm, Field Science Center for Northern Biosphere, HokkaidoUniversity. The inoculum was diluted two fold with McDougal's artificialsaliva (pH 6.8) and used.

(2) Culture

The culture was performed in such a manner that the concentration ofCNSL in a test culture medium was 500 mg/L. 0.05 g of CNSL was dissolvedin 1 ml of ethanol, and 100 μl of the solution was added to a Hungatetube. Ethanol was volatilized by leaving the resultant standing forseveral hours. Then, as a culture substrate, added therein were 0.15 gof corn starch, 0.025 g of blended feed powder, and 0.025 g of powderedorchard grass hay. 10 ml of the above-mentioned diluted rumen juice wereadded to the mixture, and the tube was sealed with a butyl rubber capand a plastic screw cap while blowing nitrogen gas to the headspace, tobe subjected to anaerobic culture in a water bath (37° C., 18 hours).

Treatments include an additive-free (ethanol only: control) zone and aCNSL zone, and the culture was performed in quintuplicate in each zone.

(3) Analysis

Methane, hydrogen, and carbon dioxide were analyzed by a TCD gaschromatography. The concentration and composition of total volatilefatty acids (VFA) were determined by FID gas chromatography.

(4) Results

(i) Gas Production

Table 1 shows the results of gas production.

Carbon dioxide and methane were decreased significantly by adding CNSL.In particular, methane was hardly detected (98% suppression). Theaccumulation of hydrogen accompanied by the decrease in methane was notobserved, and hydrogen produced in anaerobic fermentation was smoothlytransferred to an alternative hydrogen consuming system.

TABLE 1 Gas production in vitro from rumen culture medium added withCNSL CO₂ (ml) CH₄ (ml) H₂ (ml) Control zone 2.54 ± 0.65_(a) 0.78 ±0.27_(a) 0.007 ± 0.002 CNSL-added zone 1.82 ± 0.30_(b) 0.01 ± 0.01_(b)0.013 ± 0.006 CNSL was added at a final concentration of 500 mg/L.Culture was performed in quintuplicate anaerobically at 37° C. for 18hours.Significant differences are present between different alphabeticalletters.

(ii) Production of Volatile Fatty Acids (VFA)

Table 2 shows the results of the production of VFA.

The VFA concentration was not changed by adding CNSL, and thus there wasno suppression of fermentation. However, the fermentation patternchanged remarkably, acetic acid production and butyric acid productiondecreased significantly, and propionic acid production increasedsignificantly.

TABLE 2 Production of volatile fatty acids in vitro from rumen culturemedium added with CNSL Total VFA (mM/dl) Acetic acid (%) Propionic acid(%) Butyric acid (%) Control zone 7.61 ± 0.50 67.62 ± 1.78_(a) 21.59 ±0.78_(a) 9.68 ± 0.22_(a) CNSL-added zone 7.31 ± 0.73 54.75 ± 3.09_(b)40.50 ± 3.68_(b) 4.31 ± 0.50_(b) Significant differences are presentbetween different alphabetical letters. “%” refers to percentage withrespect to the total VFA.

The results link well with decrease in methane production, and propionicacid production improved remarkably as an alternative consuming systemof hydrogen.

Example 2 Antibacterial Action of CNSL

For examining the antibacterial action of CNSL, the following strainswere each cultured in a brain-heart-infusion medium (manufactured byNISSUI PHARMACEUTICAL CO., LTD.) at 37° C. for a day: Staphylococcosaureus strain isolated from a bovine; Streptococcus bovis DSM20065strain; Bacillus subtilis NBRC3009 strain; Escherichia coli ATCC11303strain; Pseudomonas aeruginosa NBRC12689 strain; and Saccharomycescerevisiae NBRC10217 strain. Into the brain-heart-infusion medium towhich CNSL was added, 10 μL each of culture media of the above-mentionedstrains were inoculated, and the resultant was cultured at 37° C. fortwo days, to thereby calculate a minimum growth-inhibitory concentration(MIC).

Table 3 shows the results.

TABLE 3 MIC(μg/ml) Gram-positive Staphylococcos aureus isolated from6.25 bacteria a bovine Streptococcus bovis DSM20065 9.38 Bacillussubtilis NBRC3009 6.25 Gram-negative Escherichia coli ATCC11303 >1600bacteria Pseudomonas aeruginosa NBRC12689 >1600 Fungus Saccharomycescerevisiae NBRC10217 >1600

CNSL does not have an antibacterial action against Gram-negativebacteria, while CNSL has a high antibacterial action againstGram-positive bacteria. That is, CNSL has a selective antimicrobialaction against rumen microorganisms.

Example 3 Effects of CNSL Concentration on Production of Methane andVolatile Fatty Acids In Vitro (1) Sample

Cashew nut shell liquid (CNSL), which was extracted by compressingcashew nut shells, was obtained from Cashew Trading Co., Ltd. For aculture inoculum, there was used rumen juice (quadruple gauze filtrate)collected from a Holstein cow (fitted with rumen cannula) owned byExperiment Farm, Field Science Center for Northern Biosphere, HokkaidoUniversity. The inoculum was diluted two fold with McDougal's artificialsaliva (pH 6.8) and used.

(2) Culture

The culture was performed in such a manner that the concentrations ofCNSL in test culture media were 50, 100, 250, and 500 mg/L. CNSL wasdissolved in 1 ml of ethanol, and 100 ml of the solution was added to aHungate tube. Ethanol was volatilized by leaving the resultant standingfor several hours. Then, as a culture substrate, added therein were 0.15g of corn starch, 0.025 g of blended feed powder, and 0.025 g ofpowdered orchard grass hay. 10 ml of the above-mentioned diluted rumenjuice were added to the mixture, and the tube was sealed with a butylrubber cap and a plastic screw cap while blowing nitrogen gas to theheadspace, to be subjected to anaerobic culture in a water bath (37° C.,18 hours).

Treatments include an additive-free (ethanol only: control) zone and aCNSL zone, and the culture was performed in quintuplicate in each zone.

(3) Results

TABLE 4 Concentration of CNSL (mg/L) 0 50 100 250 500 Amount of methaneproduction (ml)  2.43 ± 0.30_(a)  1.88 ± 0.22_(a)  1.64 ± 0.40_(a)  0.45± 0.02_(b)  0.12 ± 0.04_(c) Total VFA (mmol) 79.94 ± 2.32_(a) 77.91 ±7.59_(abc) 80.46 ± 3.64_(a) 67.56 ± 4.39_(bc) 68.70 ± 1.81_(b) Propionicacid (mmol) 19.84 ± 0.21_(a) 20.09 ± 1.39_(a) 23.43 ± 1.04_(c) 37.63 ±2.01_(b) 38.07 ± 0.85_(b) Significant differences are present betweendifferent alphabetical letters.

With the addition of 250 mg/L or more of CNSL, methane was hardlydetected. In the zone added with 250 mg/L or more of CNSL, theconcentration of total volatile fatty acids slightly decreased, but thefermentation pattern changed remarkably. With the addition of 100 mg/Lor more of CNSL, propionic acid production increased significantly.

Example 4 Effects with Time of CNSL Concentration on Production ofVolatile Fatty Acids In Vitro (1) Sample

Cashew nut shell liquid (CNSL), which was extracted by compressingcashew nut shells, was obtained from Cashew Trading Co., Ltd. For aculture inoculum, there was used rumen juice (quadruple gauze filtrate)collected from a Holstein cow (fitted with rumen cannula) owned byExperiment Farm, Field Science Center for Northern Biosphere, HokkaidoUniversity.

(2) Test

Into a 1,000-ml fermentor, 800 ml of a culture medium in which rumenjuice and McDougal's artificial saliva (pH 6.8) were mixed at a ratio of1:1 (mass) was added, and carbon dioxide was blown thereinto to therebyestablish an anaerobic condition. The artificial saliva was continuouslyfed into the fermentor, and waste liquid was collected as a sample.Generated gas was collected in a GasPak. There was a plate having holesplaced inside the fermentor, and owing to slow up-and-down movement ofthe plate, the contents were mixed. Two nylon mesh bags each of whichcontaining a coarse feed (feed) were placed inside the fermentor at alltimes, and the older bag of the two bags was replaced with another bagonce a day. CNSL was added to the feed in such a manner that theconcentrations of CNSL in culture media were 0, 50, 100, and 200 mg/L,and the culture was performed in duplicate in each culture media. Theculture period was 21 days. The culture was performed under darkconditions at all times.

(3) Results

Table 5 shows the amount (ml) of collected gas.

TABLE 5 CNSL(mg/L) Hydrogen Methane Carbon dioxide 0 1.30 67.07 500.8450 1.76 50.34 531.11 100 4.99 38.04 577.25 200 4.36 23.91 511.18

The amount of methane was decreased by adding CNSL.

Table 6 shows the concentration (mmol/dl) of total volatile fatty acids(VFA) in the collected rumen juice.

TABLE 6 CNSL (mg/L) Day 0 50 100 200 0 10.77 11.43 11.37 10.96 3 10.8010.89 12.13 11.40 6 11.73 13.03 13.39 12.69 9 12.56 12.76 12.99 12.83 1211.70 11.89 13.80 11.70 15 10.49 11.79 11.77 10.86 18 11.11 12.17 11.8311.57 21 10.65 11.35 12.19 11.21

The total VFA concentration was not changed by adding CNSL (i.e. thereis no suppression of fermentation).

Table 7 shows the molar ratio (%) of acetic acid in the collected rumenjuice, Table 8 shows the molar ratio (%) of propionic acid in thecollected rumen juice, and Table 9 shows the molar ratio (%) of butyricacid in the collected rumen juice.

TABLE 7 Molar ratio of acetic acid CNSL (mg/L) Day 0 50 100 200 0 48.645.9 44.1 41.7 3 48.9 45.9 44.3 41.4 6 49.0 46.3 44.7 41.0 9 50.2 46.645.1 41.6 12 48.6 46.2 45.1 41.7 15 50.2 45.4 45.6 40.6 18 48.2 45.343.6 41.3 21 48.1 45.5 43.4 40.9

TABLE 8 Molar ratio of propionic acid CNSL (mg/L) Day 0 50 100 200 026.8 27.6 30.6 35.8 3 26.5 27.4 30.7 35.9 6 26.1 27.4 30.8 36.1 9 25.827.2 31.2 36.1 12 26.1 27.3 31.1 35.9 15 25.4 27.1 30.4 36.0 18 26.227.7 31.5 34.5 21 25.9 27.3 31.6 36.0

TABLE 9 Molar ratio of butyric acid CNSL (mg/L) Day 0 50 100 200 0 15.315.7 14.5 12.7 3 15.4 15.7 13.9 12.6 6 15.1 15.2 13.6 12.7 9 14.5 15.213.1 12.3 12 15.3 15.1 13.2 12.5 15 14.4 15.4 13.0 12.6 18 15.2 15. 113.1 13.2 21 14.8 15.1 13.1 12.3

With the addition of 100 mg/L of CNSL and 200 mg/L of CNSL, thefermentation pattern changed remarkably, acetic acid production andbutyric acid production decreased, and propionic acid productionincreased.

Example 5 Effects with Time of CNSL Administration In Vivo (1) Sample

Four sheep fitted with rumen cannula were each provided with a feed(concentrated feed:hay=3:7 (volume)) in an amount equivalent to 1.4 mass% of the weight of the each sheep.

A first sampling of rumen contents was performed before starting theadministration of CNSL. As for the dose of CNSL, rumenfunction-improving effects were observed with the addition of 100 mg/Lor more of CNSL in the test in vitro. In order to allow theconcentration of CNSL in the rumen juice of the sheep to be 100 mg/L, itis required to mix 0.14 to 0.28 mass % (1,400 to 2,800 mass ppm) of CNSLinto the feed, because CNSL is diluted in the rumen juice. Accordingly,0.14 mass % of CNSL was added to the feed for the first two weeks andthe sampling of rumen contents was performed once a week, i.e., twice intotal. 0.28 mass % of CNSL was added to the feed for the next two weeksand the sampling of rumen contents was performed once a week, i.e.,twice in total. For the next two weeks, only a feed in which CNSL is notadded was provided to the sheep, and the sampling of rumen contents wasperformed once a week, i.e., twice in total.

(2) Results

Table 10 shows the amount of the produced gas (ml/day/tube) when thecollected rumen juice was sealed in a test tube and cultured at 37° C.for 24 hours.

TABLE 10 Carbon CNSL dose Hydrogen Methane dioxide Before starting —0.03 ± 0.00 0.83 ± 0.42 3.44 ± 0.79 administration First week 0.14 mass% 0.04 ± 0.02 0.69 ± 0.12 3.43 ± 0.79 Second week 0.14 mass % 0.03 ±0.01 0.46 ± 0.18 2.36 ± 0.72* Third week 0.28 mass % 0.07 ± 0.07 0.22 ±0.13** 1.63 ± 0.57** Fourth week 0.28 mass % 0.04 ± 0.01 0.32 ± 0.23*2.65 ± 0.90 Fifth week Discontinuation 0.02 ± 0.00** 0.44 ± 0.20 2.76 ±0.74 of administration Sixth week Discontinuation 0.03 ± 0.01* 0.73 ±0.80 3.30 ± 2.26 of administration *P < 0.10 compared to before startingadministration **P < 0.05 compared to before starting administration

The amount of methane was decreased significantly by adding CNSL. Theaccumulation of hydrogen accompanied by the decrease in methane was notobserved, and hence, it is considered that hydrogen produced inanaerobic fermentation was smoothly transferred to an alternativehydrogen consuming system.

Table 11 shows the total VFA concentration (mmol/dl), the molar ratio(%) of acetic acid, the molar ratio (%) of propionic acid, the molarratio (%) of butyric acid in the collected rumen juice.

TABLE 11 VFA Acetic Propionic Butyric CNSL dose concentration acid %acid % acid % Before starting — 3.35 ± 0.49 60.8 ± 4.0 20.7 ± 3.5 15.5 ±2.6 administration First week 0.14 mass % 3.53 ± 1.04 55.4 ± 2.9 24.0 ±3.0 16.5 ± 2.6 Second week 0.14 mass % 3.43 ± 0.92 55.7 ± 1.7* 23.6 ±5.9 11.8 ± 3.5 Third week 0.28 mass % 2.85 ± 1.03 46.8 ± 2.5** 31.0 ±8.6* 13.4 ± 6.0 Fourth week 0.28 mass % 3.05 ± 0.57 53.8 ± 1.6** 30.5 ±4.8** 10.0 ± 3.6 Fifth week Discontinuation 3.69 ± 0.77 59.7 ± 2.4 20.5± 5.4 13.2 ± 3.7 of administration Sixth week Discontinuation 4.10 ±0.82 62.4 ± 1.1 17.8 ± 3.6 13.3 ± 3.4 of administration *P < 0.10compared to before starting administration **P < 0.05 compared to beforestarting administration

The total VFA concentration was not changed by adding CNSL in an amountof 0.14 mass % (i.e. there is no suppression of fermentation). Further,with the addition of CNSL in an amount of 0.28 mass %, the fermentationpattern changed remarkably, acetic acid production decreasedsignificantly, and propionic acid production increased significantly.

Table 12 shows the ammonia concentration (mgN/dl) in the collected rumenjuice.

TABLE 12 Ammonia CNSL dose concentration Before starting — 22.82 ± 5.00administration First week 0.14 mass % 23.76 ± 3.81 Second week 0.14 mass% 21.71 ± 4.61 Third week 0.28 mass % 14.11 ± 6.62* Fourth week 0.28mass % 13.01 ± 7.27* Fifth week Discontinuation 26.59 ± 6.86 ofadministration Sixth week Discontinuation 28.80 ± 7.26 of administration*P < 0.10 compared to before starting administration **P < 0.05 comparedto before starting administration

With the administration of CNSL, the tendency of decrease in ammoniaconcentration was observed. The results show that proteolysis ordeamination is suppressed and feed efficiency is increased.

The results in vitro obtained in Examples 1 to 4 correlated well withthe results obtained in Example 5, which used sheep. That is, in anactual rumen, with the addition of CNSL, carbon dioxide and methanedecreased significantly, and because the accumulation of hydrogenaccompanied by the decrease in methane was not observed at that time, itis considered that hydrogen produced in anaerobic fermentation wassmoothly transferred to an alternative hydrogen consuming system.Further, the concentration of total volatile fatty acids was not changedby adding CNSL (i.e. there is no suppression of fermentation). However,the fermentation pattern changed remarkably, acetic acid productiondecreased significantly, and propionic acid production increasedsignificantly.

The results link well with decrease in methane production, and it isconsidered that propionic acid production developed smoothly as analternative consuming system of hydrogen. The above facts wereexemplified in the actual rumen of the sheep, and hence, it isconsidered that the cashew nut shell liquid enhances the utilizationefficiencies of energy and protein in domestic animals.

INDUSTRIAL APPLICABILITY

Methane produced by cattle is a loss of feed energy and is also agreenhouse gas, and therefore, it is an urgent issue to reduce methaneproduction from cattle from the viewpoints of zootechnical science andenvironmentology. By allowing a ruminant to ingest cashew nut shellliquid when breeding the ruminant, methane production can be suppressed.On the other hand, propionic acid has the highest transformationefficiency of feed hexose energy among the volatile fatty acids and isan original substance of sugar to be changed into glucose afterabsorption, and hence, promotion of propionic acid production leads tosaving of other original substances of sugar (e.g., amino acid). Thus,the feed containing cashew nut shell liquid can enhance the utilizationefficiencies of energy and protein in domestic animals.

1. A rumen fermentation improving agent, comprising cashew nut shellliquid.
 2. A composition for a feed for improving rumen fermentation,comprising the rumen fermentation improving agent according to claim 1.3. A feed for improving rumen fermentation, comprising the compositionfor a feed for improving rumen fermentation according to claim
 2. 4. Acomposition for a feed for improving rumen fermentation, comprisingcashew nut shell liquid.
 5. A feed for improving rumen fermentation,comprising cashew nut shell liquid.
 6. The feed for improving rumenfermentation according to claim 3, which is for a ruminant.
 7. A methodof breeding a ruminant, comprising allowing the ruminant to ingest thefeed for improving rumen fermentation according to claim
 3. 8. A methodof manufacturing a rumen fermentation improving agent comprising usingcashew nut shell liquid.
 9. A method of manufacturing a composition fora feed for improving rumen fermentation comprising using cashew nutshell liquid.
 10. A method of manufacturing a feed for improving rumenfermentation comprising incorporating cashew nut shell liquid.
 11. Amethod of improving rumen fermentation of a domestic animal, comprisingadministering cashew nut shell liquid to the domestic animal.